50 / 2021-08-05 19:11:11

Molecular docking analysis of interaction between CaM-F142L and the CaV1.2 channel

Calmodulin mutant; Molecular docking; CaV1.2 channel; Long QT syndrome

It is reported that calmodulin (CaM) mutations are associated with severe long QT syndrome (LQTS), and induced a weakening of Ca²⁺-dependent inactivation (CDI) of Ca_V1.2 channel. Long QT syndrome (LQTS) is an hereditary or acquired disease. It is clinically manifested as cardiac dysfunction, occasional recurrent syncope and epilepsy, arrhythmia, and even sudden death. CaM-F142L is a critical missense variant associated with LQTS, which located in EF-hand IV region of CaM. However, the interaction of this CaM mutant with the Ca_V1.2 channel has not been determined. In our previous study, we have already found that CaM-WT can bind with N terminus (NT) and C terminus (CT) of cardiac Ca_V1.2 channel under different Ca²⁺ concentration. In this study, we first set up the the three-dimensional structure of CaM-F142L using the I-TASSER protein structure and function prediction server, and then try to explore the interaction between CaM-F142L and the C-terminus and N-terminus of cardiac Ca_V1.2 channel (PreIQ, IQ, PreIQ-IQ, CT1, NT) by molecular docking method. Finally, the interaction results were verified by utilizing a semiquantitative pull-down assay. The results showed that the three-dimensional structure of CaM-F142L was successfully constructed, and it still have the ability to bind with PreIQ, IQ, PreIQ-IQ, CT1 and NT. In addition, we compared the conformation changes of the binding of the wild type CaM and the mutant CaM with the Ca_V1.2 channel. The binding conformation of CaM-F142L with the Ca_V1.2 channel were different from those of CaM, and the binding site also changed. The docking results were verified by GST pull-down method, which CaM-F142L can bind with PreIQ/IQ/PreIQ-IQ/CT1/NT at different Ca²⁺ concentration (≈free, 2 mM, 10 μM, 100 nM) with a protein-concentration dependent manner. These data imply that the identified LQTS-associated variants CaM-F142L disrupts the binding with cardiac Ca_V1.2 channel and provide possible therapeutic targets and ideas for treat of CaM mutant related cardiovascular diseases.